Biological fluids such as blood plasma or serum, milk whey, urine, etc. contain a mixture of several proteins. For example, blood plasma contains albumin (3.5-4.5 g/100 ml, M. wt 66,000), fibrinogen (0.20-0.45 g/100 ml, M. wt 340,000), .alpha.-globulins (0.4-1.0 g/100 ml) .beta.-globulins (0.8-1.8 g/100 ml, M. wt 160,000), IgM (0.06-0.25 g/100 ml, M. wt. 950,000), etc. (Frank W. Putnam, The Trace Components of Plasma, An Overview). The immunoglobulins (Ig's) are very important since they are involved in the protective and defensive mechanisms against infectious organisms. Clinical diseases characterized by imbalances of these systems of proteins for example either in the ability to recognize invading organisms or to recognize indigenous protein or polynucleic acids, have promoted the basic understanding of the clinical aspects of the science of immunity. Abnormal immunological reactions are now known to cause a wide spectrum of diseases. Examples of diseases known to be associated with immune complex reactions include, for example, serum sickness, flomerulonephritis and myasthenia gravis. Plasmapheresis is a technique used to curtail, favorable interfere with or stop the immunopathologic process associated with circulating humoral antibody and/or immune complexes of the plasma. [Glassman, Rationale for Plasmapheresis, "Plasma Therapy" Vol. 1 No. 1, Page 13 (1979).]
A known method is to plasmapherese about 4 liters of blood by centrifugation or cross-flow filtration over a period of 2-4 hours. The plasma removed from the patient in this way is usually discarded and replaced by albumin and either physiological saline or Ringer's solution to make up the protein, electrolyte, and water balance. This is an expensive method. In another method the replacement of the removed plasma is accomplished by giving fresh or frozen pool plasma, and though less expensive, suffers from the risk of transmitting hepatitis virus to the patient. The method of the present invention (referred to as immunepheresis) overcomes these problems by selectively removing euglobulins or euglobulin antigen complexes causing or resulting from the disease and at the same time restoring the major portions of albumin, electrolyte (salt) and water and thus returning to the patient his or her own plasma (substantially depleted in Ig or Ig antigen complex) containing the proper protein, risk free from hepatitis since no additional albumin or donor plasma is required.
Antihemophilic factor (AHF) or antihemophilic globulin (Factor VIII, AHF or AHG) is one or the constituents involved in the coagulation of blood. A hereditary disorder or blood coagulation, hemophilia, results in profuse bleeding in joints, muscles or internal organs as a result or minor trauma. This disease appears to be due to a deficiency of a specific plasma protein AHF. Affected individuals frequently require therapy following minor accidents. In case surgery is required, clotting abnormality is corrected by fresh plasma transfusions or by injection of Factor VIII concentrate, the latter being preferred since it avoids hyperproteinemia and possible kidney dysfunction resulting from large volume transfusions.
Prior art methods for production of AHF consist for example, of taking pool-plasma, forming a cryoprecipitate, centrifuging the precipitate which mainly consists of a mixture of AHF and fibrinogen, removing fibrinogen and thereafter employing lyophilization to produce AHF concentrate. These methods suffer from the disadvantages of being long and cumbersome and of having the risk of transmitting hepatitis because of the pool-plasma source. Also the presence of fibrinogen as an impurity makes it difficult for the AHF concentrates to go into solution. In addition, due to an elapse of several days between donation and use there is a considerable loss of AHF activity. An AHF unit is defined as the activity present in 1 ml. of average normal pooled human plasma which is less than 1 hour old (100% AHF level). Thus after six hours the loss in activity in extra corporeal liquid plasma can be as great as 80%. A rapid method of processing AHF would prevent this loss of activity. The apparatus and methods of the present invention overcome these problems by being suited to an on-line real-time method. Therefore the recovery of AHF can be as high as 4 to 5 times that of the present, long elapsed time methods. The present invention is adaptable to a smaller pooled source. e.g. 2-3 hepatitis-free members of the hemophiliac's family can donate plasma and have the AHF recovered on site with a short time thereafter thus providing a hepatitis free AHF of very high activity. On-line methods of this invention can also be used to recover Factor VIII from donors during plasmapheresis.
The basic techniques employed in the present invention i.e., plasmapheresis, salting-out and dialysis when combined in a novel manner as described herein produces a synergism, i.e., it increases the usefulness of each step and the combination of steps in a previously unexpected manner and makes them extremely efficacious especially for in situ therapeutic use for plasmapheresis patients where removal of Ig's or their complexes is required.
The methods of the present invention will be described using plasma proteins as the preferred examples but the scope of this invention can also be applied to other biological fluids or proteins without limiting the scope of the invention. These methods for protein separation can serve as a very efficient tool in the hands of protein chemists.